Intranuclear Positions of HIV-1 Proviruses Are Dynamic and Do Not Correlate with Transcriptional Activity

HIV-1 integrates its genomic DNA into the chromosomes of the infected cell, but how it selects the site of integration and the impact of their location in the 3-dimensional nuclear space is not well understood. Here, we examined the nuclear locations of proviruses 1 and 5 days after infection and found that integration sites are first located near the nuclear envelope but become randomly distributed throughout the nucleus after a few cell divisions, indicating that the locations of the chromosomal sites of integration that harbor transcriptionally active proviruses are dynamic.

Cucumber Mosaic Virus Infection in Arabidopsis: A Conditional Mutualistic Symbiont?

A cucumber mosaic virus isolate, named Ho [CMV(Ho)], was isolated from a symptomless Arabidopsis halleri field sample containing low virus titers. An analysis of CMV(Ho) RNA molecules indicated that the virus isolate, besides the usual cucumovirus tripartite RNA genome, additionally contained defective RNA3 molecules and a satellite RNA. To study the underlying mechanism of the persistent CMV(Ho) infection in perennial A. halleri, infectious cDNA clones were generated for all its genetic elements. CMV, which consists of synthetic transcripts from the infectious tripartite RNA genomes, and designated CMV(Ho)tr, multiplied in A. halleri and annual Arabidopsis thaliana Col-0 to a similar level as the virulent strain CMV(Y), but did not induce any symptoms in them. The response of Col-0 to a series of reassortant CMVs between CMV(Ho)tr and CMV(Y) suggested that the establishment of an asymptomatic phenotype of CMV(Ho) infection was due to the 2b gene of CMV RNA2, but not due to the presence of the defective RNA3 and satellite RNA. The accumulation of CMV(Ho) 2b protein tagged with the FLAG epitope (2b.Ho-FLAG) in 2b.Ho-FLAG-transformed Col-0 did not induce any symptoms, suggesting a 2b-dependent persistency of CMV(Ho)tr infection in Arabidopsis. The 2b protein interacted with Argonaute 4, which is known to regulate the cytosine methylation levels of host genomic DNA. Whole genomic bisulfite sequencing analysis of CMV(Ho)tr- and mock-inoculated Col-0 revealed that cytosine hypomethylation in the promoter regions of 82 genes, including two genes encoding transcriptional regulators (DOF1.7 and CBP1), was induced in response to CMV(Ho)tr infection. Moreover, the increased levels of hypomethylation in the promoter region of both genes, during CMV(Ho)tr infection, were correlated with the up- or down-regulation of their expression. Taken altogether, the results indicate that during persistent CMV(Ho) infection in Arabidopsis, host gene expression may be epigenetically modulated resulting from a 2b-mediated cytosine hypomethylation of host genomic DNA.

N4-acetyldeoxycytosine DNA modification marks euchromatin regions in Arabidopsis thaliana

Background Direct analogs of chemically modified bases that carry important epigenetic information, such as 5-methylcytosine (m5C)/5-methyldeoxycytosine (5mC), 5-hydroxymethylcytosine (hm5C)/5-hydroxymethyldeoxycytosine (5hmC), and N6-methyladenosine (m6A)/N6-methyldeoxyadenosine (6mA), are detected in both RNA and DNA, respectively. The modified base N4-acetylcytosine (ac4C) is well studied in RNAs, but its presence and epigenetic roles in cellular DNA have not been explored. Results Here, we demonstrate the existence of N4-acetyldeoxycytosine (4acC) in genomic DNA of Arabidopsis with multiple detection methods. Genome-wide profiling of 4acC modification reveals that 4acC peaks are mostly distributed in euchromatin regions and present in nearly half of the expressed protein-coding genes in Arabidopsis. 4acC is mainly located around transcription start sites and positively correlates with gene expression levels. Imbalance of 5mC does not directly affect 4acC modification. We also characterize the associations of 4acC with 5mC and histone modifications that cooperatively regulate gene expression. Moreover, 4acC is also detected in genomic DNA of rice, maize, mouse, and human by mass spectrometry. Conclusions Our findings reveal 4acC as a hitherto unknown DNA modification in higher eukaryotes. We identify potential interactions of this mark with other epigenetic marks in gene expression regulation.

Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae) in the host Anaphes nitens (Hymenoptera: Mymaridae): first report of association with insects

Endosymbiont bacteria can affect biological parameters and reduce the effectiveness of natural enemies in controlling the target insect. The objective of this work was to identify endosymbiont bacteria in Anaphes nitens (Girault, 1928) (Hymenoptera: Mymaridae), the main natural enemy used to manage Gonipterus platensis (Marelli, 1926) (Coleoptera: Curculionidae). Genomic DNA from six A. nitens populations was extracted and polymerase chain reactions (PCR) were performed with the primers to detect endosymbiont bacteria in this insect. The PCR products were amplified, sequenced, and compared with sequences deposited in the GenBank for the bacteria identification. All A. nitens populations had the bacterium Yersinia massiliensis (Enterobacteriales: Enterobacteriaceae). This bacterium was originally described as free-living, and it is associated with and composes part of the A. nitens microbiota. This is the first report of Y. massiliensis in an insect host.

Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

Hepatitis B virus genomic nucleic acid in the activation and maturation of bone marrow-derived dendritic cells

Hepatitis B virus (HBV) is the etiological agent that causes a self-limiting or chronic infection in the hepatocytes of about 250 million people worldwide. The role of adaptive immune system during HBV infection has been well studied. However, the innate immune system's responses against HBV during the early stage of infection largely remain unclear. In this study, we found that HBV genomic DNA or Salmon Sperm DNA (SSD) was able to induce the innate immune response in the macrophages cell line RAW264.7 but not the hepatocyte cell line, HepG2, indicating that hepatocytes may lack of a functional DNA-sensing pathway and hence are unable to respond to the presence of foreign DNA in the cytosol with type 1 IFN response. Thus, we hypothesized that non-parenchymal cells like the Antigen Presenting Cells (APC) might be crucial in triggering the initial immune response to suppress the virus replication and link the innate and adaptive responses. Using bone marrow-derived DCs (BMDC) as a model, this study demonstrated that HBV genomic DNA is able to induce cytokines like TNF-alpha, IL-6, and IL-12p40 secretion. We also examined the activation and maturation of BMDCs when exposed to the HBV genomic DNA intracellularly and extracellularly. A significant shift of CD86+ and CD40+ cell populations was observed during extracellular exposure of BMDC to Poly I:C and HBV genomic DNA, indicating that TLRs may be vital in the uptake of the extracellular viral DNA to activate the BMDCs. Moreover, transfection of intracellular nucleic acid stimuli, including HBV genomic DNA as well induced BMDCs maturation. Our findings highlight the critical function of DCs in antiviral response as a potential connection between the innate and adaptive immune systems during HBV pathogenesis. Nevertheless, further study is required to determine the role of cytosol DNA sensing pathway in DCs during HBV infection.

Along the Bos Taurus genome, uncover candidate Imprinting Control Regions

In mammals, Imprinting Control Regions (ICRs) regulate a subset of genes in a parent-of-origin-specific manner. In both human and mouse, previous studies identified a set of CpG-rich motifs that occurred as clusters in ICRs and germline Differentially Methylated Regions (gDMRs). These motifs consist of the ZFP57 binding site (ZFBS) overlapping a subset of MLL binding units known as MLL morphemes. Furthermore, by creating plots for displaying the density of these overlaps, it became possible to locate known and candidate ICRs in mouse and human genomic DNA. Since genomic imprinting impacts many developmental and key physiological processes, we performed genome-wide analyses to create plots displaying the density of the CpG-rich motifs (ZFBS-morph overlaps) along Bos Taurus chromosomal DNA. We tailored our datasets so that they could be displayed on the UCSC genome browser (the build bosTau8). On the genome browser, we could view the ZFP57 binding sites, the ZFBS-morph overlaps, and peaks in the density-plots in the context of cattle RefSeq Genes, Non-Cow RefSeq Genes, CpG islands, and Single nucleotide polymorphisms (SNPs). Our datasets revealed the correspondence of peaks in plots to known and deduced ICRs in Bos Taurus genomic DNA. We illustrate that by uploading our datasets onto the UCSC genome browser, we could discover candidate ICRs in cattle DNA. In enlarged views, we could pinpoint the genes in the vicinity of candidate ICRs and thus discover potential imprinted genes.

Whole Exome-Sequencing of Pooled Genomic DNA Samples to Detect Quantitative Trait Loci in Esotropia and Exotropia of Strabismus in Japanese

Background: Esotropia and exotropia are two major phenotypes of comitant strabismus. It remains controversial whether esotropia and exotropia would share common genetic backgrounds. In this study, we used a quantitative trait locus (QTL)-sequencing pipeline for diploid plants to screen for susceptibility loci of strabismus in whole exome sequencing of pooled genomic DNAs of individuals. Methods: Pooled genomic DNA (2.5 ng each) of 20 individuals in three groups, Japanese patients with esotropia and exotropia, and normal members in the families, was sequenced twice after exome capture, and the first and second sets of data in each group were combined to increase the read depth. The SNP index, as the ratio of variant genotype reads to all reads, and Δ(SNP index) values, as the difference of SNP index between two groups, were calculated by sliding window analysis with a 4 Mb window size and 10 kb slide size. The rows of 200 “N”s were inserted as a putative 200-b spacer between every adjoining locus to depict Δ(SNP index) plots on each chromosome. SNP positions with depth <20 as well as SNP positions with SNP index of <0.3 were excluded. Results: After the exclusion of SNPs, 12,242 SNPs in esotropia/normal group and 12,108 SNPs in exotropia/normal group remained. The patterns of the Δ(SNP index) plots on each chromosome appeared different between esotropia/normal group and exotropia/normal group. When the consecutive groups of SNPs on each chromosome were set at three patterns: SNPs in each cytogenetic band, 50 consecutive sliding SNPs, and SNPs in 4 Mb window size with 10 kb slide size, p values (Wilcoxon signed rank test) and Q values (false discovery rate) in a few loci as Manhattan plots showed significant differences in comparison between the Δ(SNP index) in the esotropia/normal group and exotropia/normal group. Conclusions: The pooled DNA sequencing and QTL mapping approach for plants could provide overview of genetic background on each chromosome and would suggest different genetic backgrounds for two major phenotypes of comitant strabismus, esotropia and exotropia.

Comparative Plasmid-Mediated Molecular Resistance Status of Diarrheic Escherichia coli Isolates from Human and Goat Kids

Globally antibiotic resistance has become a major concern, which warrants the real time monitoring for resistance in very common pathogenic organisms. E. coli is normal micro flora in humans, but sometimes it can be pathogenic. For the observation and increment of antimicrobial resistance among pathogen, E. coli has been one of the important pathogens. It is present everywhere in fecal, water, food etc., if resistant E. coli will present in the environment that it can be transferrable anywhere through water, fecal food, animals and humans. This is very dangerous to living beings. This study was designed on status of antibiotic resistance in E. coli isolates in human kids and animal kids, both. Newborns are affected more because of poor or lack of immune system. In this study, fecal materials were used as sample material collected from goat kids (0-3 months) and human children (up to 3 years) residing in same local area. Fifteen fecal samples were collected from human children (up to 3 years) and goat kids (0-3 months) in each case to study the risk of transmission of resistance in E. coli isolates. PCR was conducted on genomic DNA isolates for the presence of usp A gene of E. coli. Multiplex PCR were conducted on plasmid DNA isolates for the resistance specific genes. Molecular resistance results in goat kids isolates showed resistance to antibiotics with tetracycline, sulphonamide, gentamycin, streptomycin and cephalothin to the level of 93.33, 53.33, 46.66, 13.33 & 6.66% respectively, whereas, human E. coli isolates were showed the highest resistance to sulphonamide, Tetracycline and β-lactams were as 53.33, 46.66 and 13.33% respectively but no resistance with gentamycin and streptomycin. Here, we concluded that humans and animals both were refractory to the various groups of antibiotics. This study will help in making the strategy for prevention or reduction of resistance in public